The purpose of these studies is to establish a better understanding of the energy metabolism in tissues, in vivo. Towards this goal, the laboratory concentrates on the use of non-invasive and non-destructive techniques to evaluate the biochemical and physiological function of the heart and skeletal muscle with regard to energy metabolism. The following major findings were made over the last year: 1)Using new optical and ion selective electrodes techniques the effect of calcium ions (Ca) on the ATP synthesis pathways in isolated porcine heart mitochondria was established. It was found that Ca activates not only the dehydrogenases associated with NADH generation but also the F1-Fo-ATPase directly in intact mitochondria. The effect of Ca2+ has been shown to have adequate gain to drive oxidative phosphorylation. In addition, the kinetics of this effect are on the order of 100 msec suggesting that even beat-to-beat modulation could be possible. This regulatory action of cytosolic Ca on ATP production may be an important element in the balance of cardiac energy metabolism with workload in vivo. 2) Using confocal fluorescence microscopy the distribution of mitochondrial NADH within single cardiac myocytes has been determined. Using fluorescence photo-bleaching methods the dehydrogenase activity has been quantitated within single living cells. 3) Using confocal microscopy the topology of the metabolic response to increases in workload was evaluated in single skeletal muscle fibers from xenopus. These studies reveal a heterogeneity of the metabolic responses to a increase in workload within a single cell. The cytosolic control network responsible for this metabolic compartmentation is under investigation.